WO2003016542A1 - Genes lies a l'obesite exprimes au moins dans l'hypothalamus - Google Patents

Genes lies a l'obesite exprimes au moins dans l'hypothalamus Download PDF

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WO2003016542A1
WO2003016542A1 PCT/AU2002/001099 AU0201099W WO03016542A1 WO 2003016542 A1 WO2003016542 A1 WO 2003016542A1 AU 0201099 W AU0201099 W AU 0201099W WO 03016542 A1 WO03016542 A1 WO 03016542A1
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agt
seq
nucleic acid
nucleotide sequence
acid molecule
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PCT/AU2002/001099
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English (en)
Inventor
Greg Collier
Ken Walder
Paul Zev Zimmet
Jim Trevaskis
Janine Mcmillan
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Autogen Research Pty Ltd
International Diabetes Institute
Deakin University
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Priority claimed from AUPR7042A external-priority patent/AUPR704201A0/en
Application filed by Autogen Research Pty Ltd, International Diabetes Institute, Deakin University filed Critical Autogen Research Pty Ltd
Publication of WO2003016542A1 publication Critical patent/WO2003016542A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates generally to nucleic acid molecules expressed at least in the hypothalamus identified using differential display techniques under differing physiological conditions.
  • the nucleic acid molecules are associated with or act as markers for conditions of a healthy state, obesity, anorexia, weight maintenance, diabetes and/or metabolic energy levels. More particularly, the present invention is directed to a nucleic acid molecule and/or its expression product for use in therapeutic and diagnostic protocols for conditions such as obesity, anorexia, weight maintenance, diabetes and energy imbalance.
  • nucleic acid molecule and expression product and their derivatives, homologs, analogs and mimetics are proposed to be useful, therefore, as therapeutic and diagnostic agents for obesity, anorexia, weight maintenance, diabetes and energy imbalance or as targets for the design and/or identification of modulators of their activity and/or function.
  • Obesity is a complex and heterogeneous disorder and has been identified as a key risk indicator of preventable morbidity and mortality since obesity increases the risk of a number of other metabolic conditions including type 2 diabetes mellitus and cardiovascular disease (Must et al., JAMA. 282(16): 1523-1529, 1999; Kopelman, Nature 404: 635-643, 2000).
  • type 2 diabetes mellitus and cardiovascular disease Malt et al., JAMA. 282(16): 1523-1529, 1999; Kopelman, Nature 404: 635-643, 2000.
  • the prevalence of diabetes continues to increase rapidly. It has been estimated that there were about 700,000 persons with diabetes in Australia in 1995 while in the US, diabetes prevalence increased from 4.9% in 1990 to 6.9% in 1999 (Mokdad, Diabetes Care 24(2): 412, 2001).
  • hypothalamus A number or organs/tissues have been implicated in the pathophysiology of obesity and type 2 diabes.
  • One organ of particular interest is the hypothalamus.
  • LHA lateral hypothalamus
  • NMH ventromedial hypothalamus
  • the dual-center hypothesis has been repeatedly modified to accommodate the increasing information about the roles played by various other brain regions, neurotransmitter systems and hormonal and neural signals originating in the gut on the regulation of food intake.
  • the paraventricular nucleus (PVN) is now considered to have an important integrative function in the control of energy intake.
  • NPY neuropeptide Y
  • GLP-1 glucagon-like peptide 1
  • MCH melanin-concentrating hormone
  • GAB A ⁇ -aminobutyric acid
  • genetic sequences were sought which are differentially expressed in lean and obese animals or in fed compared to unfed animals. Novel genes are identified which are proposed to be associated with or act as markers for energy balance as well as a healthy state, obesity, anorexia, weight maintenance and diabetes.
  • SEQ ID NO: Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:).
  • the SEQ ID NOs: correspond numerically to the sequence identifiers ⁇ 400>1 (SEQ ID NO:l), ⁇ 400>2 (SEQ ID NO:2), etc.
  • a sequence listing is provided after the claims.
  • Group A lean animals (normoglycemic; normoinsulinemic);
  • Group B obese, non-diabetic animals (normoglycemic; hyperinsulinemic); and
  • Group C obese, diabetic animals (hyperglycemic; hyperinsulinemic).
  • mice were maintained under fed or unfed conditions or under conditions of high or low glucose or insulin and genetic sequences analyzed by differential display analysis.
  • three differentially expressed sequences were identified from hypothalamus cells designated herein AGT-105, AGT-111 and AGT- 112 with sequence identifiers SEQ ID NO:l, SEQ ID NO:2 and SEQ ID NO:3, respectively.
  • Differential expression means an elevation in levels of expression of a genetic sequence under one set of conditions compared to another.
  • AGT-105 expression was elevated in the hypothalamus of Group A animals compared to Group B or C animals.
  • AGT-111 gene expression was elevated in the hypothalamus under fasting conditions in Group A, B and C animals.
  • AGT-112 gene expression was elevated in the hypothalamus of Group B animals.
  • AGT-105 is expressed higher in health animals versus obese, diabetic/non-diabetic rats.
  • AGT-111 expression is higher in all types of animals under fasting conditions.
  • AGT-112 is elevated in obese, non-diabetic rats under fasting conditions.
  • Table 1 A summary of the AGT sequences is provided in Table 1.
  • the identification of these variably expressed sequences permits the rationale design and/or selection of molecules capable of antagonizing or agonizing the expression products and/or permits the development of screening assays.
  • the screening assays include assessing the physiological status of a particular subject.
  • one aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a protein or mRNA or a derivative, homolog, analog or mimetic thereof wherein the nucleic acid molecule is expressed in larger amounts in hypothalamus of fasted animals compared to fed animals.
  • the nucleic acid molecule comprises a nucleotide sequence substantially as set forth in SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:3 or a nucleotide sequence having at least about 30% similarity to all or part of SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:3 and/or is capable of hybridizing to one or more of SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:3 or their complementary forms under low stringency conditions at 42°C.
  • Another aspect of the present invention provides an isolated molecule or a derivative, homolog, analog or mimetic thereof which is produced in a larger amount in hypothalamus tissue of obese animals compared to lean animals and/or which is produced in a larger amount in hypothalamus tissue of fasted animals compared to fed animals.
  • the molecule is generally a protein but may also be an mRNA, intron or exon. In this respect, the molecule may be considered an expression product of the subject nucleotide sequences.
  • the nucleic acid molecule comprises a nucleotide sequence substantially set forth in SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:3 or a nucleotide sequence having at least about 30% similarity to all or part of SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:3 and/or is capable of hybridizing to one or more of SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO:3 or their complementary forms under low stringency conditions.
  • the preferred genetic sequence of the present invention are referred to herein as AGT-105, AGT-111 and AGT-112.
  • the expression products encoded by AGT-105, AGT-111 and AGT-112 are referred to herein as AGT-105, AGT-111 and AGT-112, respectively.
  • the expression product may be an RNA (e.g. mRNA) or a protein. Where the expression product is an RNA, the present invention extends to RNA-related molecules associated thereto such as RNAi.
  • a further aspect of the present invention relates to a composition
  • a composition comprising AGT-105, AGT-111 and/or AGT-112 or its derivatives, homologs, analogs or mimetics or agonists or antagonists of AGT-105, AGT-111 and/or AGT-112 together with one or more pharmaceutically acceptable carriers and/or diluents.
  • Yet a further aspect of the present invention contemplates a method for treating a subject comprising administering to said subject a treatment effective amount of AGT-105, AGT- 111 and/or AGT-112 or a derivative, homolog, analog or mimetic thereof or a genetic sequence encoding same or an agonist or antagonist of AGT-105, AGT-111 and/or AGT- 112 activity or AGT-105, AGT-111 and/or AGT-112 gene expression for a time and under conditions sufficient to effect treatment.
  • treatments contemplated herein include but are not limited to obesity, anorexia, weight maintenance, energy imbalance and diabetes. Treatment may be by the administration of a pharmaceutical composition or genetic sequences via gene therapy. Treatment is contemplated for human subjects as well as animals such as animals important to livestock industry.
  • Still yet another aspect of the present invention is directed to a diagnostic agent for use in monitoring or diagnosing conditions such as but not limited to obesity, anorexia, weight maintenance, energy imbalance and/or diabetes, said diagnostic agent selected from an antibody to AGT-105, AGT-111 or AGT-112 or its derivatives, homologs, analogs or mimetics and a genetic sequence comprising or capable of annealing to a nucleotide strand associated with AGT-105, AGT-111 or AGT-112 useful inter alia in PCR, hybridization and/or RFLP.
  • a diagnostic agent for use in monitoring or diagnosing conditions such as but not limited to obesity, anorexia, weight maintenance, energy imbalance and/or diabetes
  • said diagnostic agent selected from an antibody to AGT-105, AGT-111 or AGT-112 or its derivatives, homologs, analogs or mimetics and a genetic sequence comprising or capable of annealing to a nucleotide strand associated with AGT-105, AGT-111 or AGT-11
  • Figure 1 is a graphical representation showing AGT-105 gene expression in Group A, B and C Psammomys obesus animals.
  • Figures 2A, 2B, 2C and 2D are graphical representations o ⁇ AGT-105 gene expression: (A) body weight in all animals; (B) versus body weight in Group A animals; (C) versus percent body fat in all animals; (D) versus log insulin in all animals (reference to "all animals” means in Group A, B and C animals).
  • Figures 3 A, 3B, 3C and 3D are graphical representations o ⁇ AGT-105 gene expression in Group A, B and C Psammomys obesus in (A) fed or fasted animals' hypothalamus on group basis; (B) in fed or fasted animals' hypothalamus on total animal basis; (C) versus log insulin in fasted animals.
  • Figure 4 is a representation of the nucleotide sequence o ⁇ Psammomys obesus AGT-105 cDNA.
  • Figures 5A and 5B are graphical representations o ⁇ AGT-111 gene expression in Group A, B and C Psammomys obesus in (A) fed or fasted animals in hypothalamus on Group basis; (B) in fed or fasted animals' hypothalamus on total animal basis.
  • Figures 6 A, 6B, 6C and 6D are graphical representations o ⁇ AGT-111 gene expression in hypothalamus (A) versus log glucose (glu) in all animals; (B) versus log glucose in fasted animals; (C) versus log glucose in fed animals; and (D) versus log insulin (ins) in all animals.
  • Figure 7 is a representation of the nucleotide sequence o ⁇ Psammomys obesus AGT-111 cDNA.
  • Figures 8A and 8B are graphical representations of AGT-112 gene expression in Psammomys obesus in Group A, B and C animals in (A) fed or fasted animals in hypothalamus on Group basis; (B) in fed or fasted animals' hypothalamus on total animal basis.
  • Figures 9A and 9B are graphical representations of AGT-112 gene expression in hypothalamus (A) versus log glucose in all animals; (B) versus log glucose in fasted animals.
  • the present invention is predicated in part on the identification of novel genes associated ter alia with regulation of energy balance, obesity and diabetes.
  • the genes were identified following differential screening of hypothalamus mRNA between lean and obese animals and/or between fed animals and fasted animals.
  • differential array is used in its broadest sense to include the expression of nucleic acid sequences in one type of tissue relative to another type of tissue in the same or different animals.
  • Reference to "different” animals include the same animals but in different gastronomical states such as in a fed or non-fed state.
  • one aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an expression product or a derivative, homolog, analog or mimetic thereof wherein said nucleic acid molecule is expressed in larger amounts in hypothalamus tissue of fasted animals compared to fed animals.
  • nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an expression product or a derivative, homolog, analog or mimetic thereof wherein said nucleic acid molecule is expressed in larger amounts in hypothalamus tissue of fasted animals compared to fed animals.
  • the elevated expression levels may be in healthy animals or in obese, diabetic or non- diabetic animals.
  • lean and "obese” are used in their most general sense but should be considered relative to the standard criteria for determining obesity. Generally, for human subjects, the definition of obesity is BMI>30 (Risk Factor Prevalence Study Management
  • an animal model may be employed to study the differences in gene expression between obese and lean animals and fasted and fed animals.
  • the present invention is exemplified using the Psammomys obesus (the Israeli sand rat) animal model of dietary-induced obesity and NIDDM.
  • Psammomys obesus the Israeli sand rat
  • an active lifestyle and saltbush diet ensure that they remain lean and normoglycemic (Shafrir and Gutman, J Basic Clin Physiol Pharm 4: 83-99, 1993).
  • Psammomys obesus exhibit a range of bodyweight and blood glucose and insulin levels which forms a continuous curve that closely resembles the patterns found in human populations, including the inverted U-shaped relationship between blood glucose and insulin levels known as "Starling's curve of the pancreas" (Barnett et al, [1994a; supra]). It is the heterogeneity of the phenotypic response o ⁇ Psammomys obesus which make it an ideal model to study the etiology and pathophysiology of obesity and NIDDM.
  • Psammomys obesus animals are conveniently divided into three groups viz Group A animals which are lean, normoglycemic and normoinsulinemic, Group B animals which are obese, normoglycemic and hyperinuslinemic and Group C animals which are obese, hyperglycemic and h yperinsulinemic.
  • nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding an expression product or a derivative, homolog, analog or mimetic thereof wherein said nucleotide sequence is as substantially set forth in SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO:3 or a nucleotide sequence having at least about 30% similarity to all or part of SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:3 and/or is capable of hybridizing to one or more of SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:3 or their complementary forms under low stringency conditions and wherein said nucleic acid molecule is expressed in a larger amount in hypothalamus tissue of obese animals compared to lean animals and/or in fed animals compared to fasted animals.
  • An expression product includes an RNA molecule such as a mRNA transcript as well as a protein.
  • Some genes are non-protein encoding genes and produce mRNA or other RNA type molecules and are involved in regulation by RNA:DNA, RNA:RNA or RNA:protein interaction.
  • the RNA e.g. mRNA
  • the RNA may act directly or via the induction of other molecules such as RNAi or via products mediated from splicing events (e.g. exons or introns).
  • Other genes encode mRNA transcripts which are then translated into proteins.
  • a protein includes a polypeptide.
  • the differentially expressed nucleic acid molecules therefore, may encode niRNAs only or, in addition, proteins. Both mRNAs and proteins are forms of "expression products".
  • Reference herein to similarity is generally at a level of comparison of at least 15 consecutive or substantially consecutive nucleotides.
  • similarity includes exact identity between compared sequences at the nucleotide level. Where there is non-identity at the nucleotide level, "similarity” includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide sequence comparisons are made at the level of identity rather than similarity.
  • sequence relationships between two or more polynucleotides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”.
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units in length. Because two polynucleotides may each comprise (1) a sequence (i.e.
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence.
  • the comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al.
  • Altschul et al. Nucl Acids Res. 25: 3389, 1997.
  • a detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, 1994-1998, Chapter 15).
  • sequence similarity and “sequence identity” as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by- nucleotide basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • sequence identity will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.
  • Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions.
  • low stringency is at from about 25-30°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions.
  • Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions.
  • medium stringency which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions
  • high stringency which includes and encompasses from at least about 31% v/v to at least about 50% v/v form
  • T m of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey, Ewr. J. Biochem. 46: 83, 1974.
  • Formamide is optional in these hybridization conditions.
  • particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42°C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65°C.
  • the nucleotide sequence or amino acid sequence of the present invention may correspond to exactly the same sequence of the naturally occurring gene (or corresponding cDNA) or protein or other expression product or may carry one or more nucleotide or amino acid substitutions, additions and/or deletions.
  • the nucleotide sequences set forth in SEQ ID NO:l, SEQ ID NO:2 and SEQ ID NO:3 correspond to novel genes referred to herein as AGT-105, AGT-111 and AGT-112, respectively.
  • the corresponding expression products are AGT- 105 , AGT- 111 and AGT- 112, respectively.
  • references herein to A GT-105, A GT- 111 and AGT-112 includes, where appropriate, reference to the genomic gene or cDNA as well as any naturally occurring or induced derivatives. Apart from the substitutions, deletions and/or additions to the nucleotide sequence, the present invention further encompasses mutants, fragments, parts and portions of the nucleotide sequence corresponding to AGT-105, AGT-111 and AGT-112.
  • nucleic acid molecule or derivative, homolog or analog thereof comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in SEQ ID NO: 1 or a derivative, homolog or mimetic thereof or having at least about 30% similarity to all or part of SEQ ID NO:l.
  • nucleic acid molecule or derivative, homolog or analog thereof comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in SEQ ID NO:2 or a derivative, homolog or mimetic thereof or having at least about 30% similarity to all or part of SEQ ID NO:2.
  • Still yet another aspect of the present invention provides a nucleic acid molecule or derivative, homolog or analog thereof comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in SEQ ID NO: 3 or a derivative, homolog or mimetic thereof or having at least about 30% similarity to all or part of SEQ ID NO:3.
  • the expression pattern o ⁇ AGT-105, AGT-111 and AGT-112 has been determined, ter alia, to indicate an involvement in the regulation of one or more of obesity, anorexia, weight maintenance, diabetes and/or energy metabolism.
  • these genes may also be expressed in other tissues including but in no way limited to brain stem, cerebellum, cortex, hippocampus and mid-brain.
  • the nucleic acid molecule encoding each of AGT-105, AGT-111 or AGT-112 is preferably a sequence of deoxyribonucleic acids such as a cDNA sequence or a genomic sequence.
  • a genomic sequence may also comprise exons and introns.
  • a genomic sequence may also include a promoter region or other regulatory regions.
  • a homolog is considered to be a gene from another animal species which has the same or greater than 30% similarity to one o ⁇ AGT-105, AGT-111 or AGT-112 and/or which has a similar function.
  • the AGT-105, AGT-111 and AG5-112 genes are exemplified herein from Psammomys obesus hypothalamus.
  • the present invention extends, however, to the homologous gene, as determined by nucleotide sequence and/or function, from humans, primates, livestock animals (e.g. cows, sheep, pigs, horses, donkeys), laboratory test animals (e.g. mice, guinea pigs, hamsters, rabbits), companion animals (e.g. cats, dogs) and captured wild animals (e.g. rodents, foxes, deer, kangaroos).
  • livestock animals e.g. cows, sheep, pigs, horses, donkeys
  • laboratory test animals e.g.
  • the nucleic acids of the present invention and in particular AGT-105, AGT-111 and AGT- 112 and their derivatives and homologs may be in isolated or purified form and/or may be ligated to a vector such as an expression vector.
  • Expression may be in a eukaryotic cell line (e.g. mammalian, insect or yeast cells) or in microbial cells (e.g. E. coli) or both.
  • isolated is meant a nucleic acid molecule having undergone at least one purification step and this is conveniently defined, for example, by a composition comprising at least about 10% subject nucleic acid molecule, preferably at least about 20%, more preferably at least about 30%, still more preferably at least about 40-50%, even still more preferably at least about 60-70%, yet even still more preferably 80-90% or greater of subject nucleic acid molecule relative to other components as determined by molecular weight, encoding activity, nucleotide sequence, base composition or other convenient means.
  • the nucleic acid molecule of the present invention may also be considered, in a preferred embodiment, to be biologically pure.
  • the nucleic acid molecule may be ligated to an expression vector capable of expression in a prokaryotic cell (e.g. E. coli) or a eukaryotic cell (e.g. yeast cells, fungal cells, insect cells, mammalian cells or plant cells).
  • the nucleic acid molecule may be ligated or fused or otherwise associated with a nucleic acid molecule encoding another entity such as, for example, a signal peptide. It may also comprise additional nucleotide sequence information fused, linked or otherwise associated with it either at the 3' or 5' terminal portions or at both the 3' and 5' terminal portions.
  • the nucleic acid molecule may also be part of a vector, such as an expression vector.
  • the derivatives of the nucleic acid molecule of the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in co- suppression and fusion nucleic acid molecules.
  • Ribozymes and DNAzymes are also contemplated by the present invention directed to AGT-105, AGT-111 and AGT-112 or their mRNAs.
  • Derivatives and homologs of AGT-105, AGT-111 and AGT-112 are conveniently encompassed by those nucleotide sequences capable of hybridizing to one or more of S ⁇ Q ID NOT, S ⁇ Q ID NO:2 or S ⁇ Q ID NO:3 or their complementary forms under low stringency conditions.
  • Derivatives include fragments, parts, portions, mutants, variants and mimetics from natural, synthetic or recombinant sources including fusion nucleic acid molecules. Derivatives may be derived from insertion, deletion or substitution of nucleotides.
  • Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids.
  • Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in a protein although random insertion is also possible with suitable screening of the resulting product.
  • Deletional variants are characterized by the removal of one or more amino acids from the sequence.
  • Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place.
  • substitutional amino acid variants are conservative amino acid substitutions.
  • Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
  • Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins.
  • Chemical and functional equivalents of protein forms of the expression products AGT-105, AGT-111, AGT-112 should be understood as molecules exhibiting any one or more of the functional activities of these molecules and may be derived from any source such as being chemically synthesized or identified via screening processes such as natural product screening.
  • the derivatives include fragments having particular epitopes or parts of the entire protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules.
  • AGT-105, AGT-111 or AGT-112 includes reference to isolated or purified naturally occurring AGT-105, AGT-111 or AGT-112 as well as any derivatives, homologs, analogs and mimetics thereof. Derivatives include parts, fragments and portions of AGT-105, AGT-111 and AGT-112 as well as single and multiple amino acid substitutions, deletions and/or additions to AGT-105, AGT-111 and AGT-112 when the expression products are proteins.
  • a derivative of AGT-105, AGT-111 or AGT-112 is conveniently encompassed by molecules encoded by a nucleotide sequence capable of hybridizing to SEQ ID NOT, SEQ ID NO:2 or SEQ ID NO:3 under low stringency conditions.
  • AGT-105, AGT-111 and AGT-105 include chemical analogs.
  • Analogs of AGT-105, AGT-111 and AGT-112 contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose confirmational constraints on the proteinaceous molecule or their analogs.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH 4 .
  • modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS);
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenyhnercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3 -nitro tyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, orni thine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • a list of unnatural amino acid, contemplated herein is shown in Table 3.
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • D-cysteine Dcys L-N-methylnorleucine Nmnle
  • D-glutamine Dgln L-N-methylnorvaline Nmnva
  • peptides can be conformationally constrained by, for example, incorporation of C ⁇ and N ⁇ -methylamino acids, introduction of double bonds between C a and C ⁇ atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
  • the expression product may be an RNA or protein.
  • protein should be understood to encompass peptides, polypeptides and proteins.
  • the protein may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • Reference hereinafter to a "protein” includes a protein comprising a sequence of amino acids as well as a protein associated with other molecules such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • the expression product is encoded by a sequence of nucleotides as set forth in SEQ ID NOT or a derivative, homolog or analog thereof including a nucleotide sequence having at least about 30% similarity to SEQ ID NOT.
  • the expression product is encoded by a sequence of nucleotides as set forth in SEQ ID NO:2 or a derivative, homolog or analog thereof including a nucleotide sequence having at least about 30% similarity to SEQ ID NO:2.
  • the expression product is encoded by a sequence of nucleotides as set forth in SEQ ID NO:3 or a derivative homolog or analog thereof including a nucleotide sequence having at least about 30% similarity to SEQ ID NO:3.
  • Another aspect of the present invention is directed to an isolated expression product selected from the list consisting of:-
  • a protein encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence as set forth in SEQ ID NOT or its complementary form or a derivative, homolog or analog thereof under low stringency conditions;
  • (x) a protein encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence as set forth in SEQ ID NO: 3 or its complementary form or a derivative, homolog or analog thereof under low stringency conditions.
  • the protein of the present invention is preferably in isolated form.
  • isolated is meant a protein having undergone at least one purification step and this is conveniently defined, for example, by a composition comprising at least about 10% subject protein, preferably at least about 20%, more preferably at least about 30%, still more preferably at least about 40-50%, even still more preferably at least about 60-70%, yet even still more preferably 80-90% or greater of subject protein relative to other components as determined by molecular weight, amino acid sequence or other convenient means.
  • the protein of the present invention may also be considered, in a preferred embodiment, to be biologically pure.
  • AGT-105, AGT-111 and/or AGT-112 is thought to relate to regulation of body weight and glucose homeostasis. Modulation of these genes expression is thought ter alia to regulate energy balance via effects on energy intake and also effects on carbohydrate/fat metabolism. The energy intake effects are likely to be mediated via the central nervous system but peripheral effects on the metabolism of both carbohydrate and fat are possible.
  • the expression of these genes may also be regulated by fasting and feeding. Accordingly, regulating the expression and/or activity of these genes or their expression products provides a mechanism for regulating both body weight and energy metabolism, including carbohydrate and fat metabolism.
  • the identification o ⁇ AGT-105, AGT-111 and AGT-112 permits the generation of a range of therapeutic molecules capable of modulating expression of AGT-105, AGT-111 and AGT-112 or modulating the activity of AGT-105, AGT-111 and AGT-112.
  • Modulators contemplated by the present invention include agonists and antagonists o ⁇ AGT-105, AGT- 111 and AGT-112 expression.
  • Antagonists o ⁇ AGT-105, AGT-111 and AGT-112 expression include antisense molecules, ribozymes and co-suppression molecules including RNAi- type molecules.
  • Agonists include molecules which increase promoter activity or which interfere with negative regulatory mechanisms.
  • Antagonists of AGT-105, AGT-111 and AGT-112 include antibodies and inhibitor peptide fragments. All such molecules may first need to be modified to enable such molecules to penetrate cell membranes. Alternatively, viral agents may be employed to introduce genetic elements to modulate expression of AGT-105, AGT-111 and AGT-112. Insofar as AGT-105, AGT-111 and AGT-112 act in association with other genes such as the ob gene which encodes leptin, the therapeutic molecules may target AGT-105, AGT-111 and AGT-112 and ob genes or their translation products.
  • the present invention contemplates, therefore, a method for modulating expression of AGT-105, AGT-111 and AGT-112 in a mammal, said method comprising contacting the AGT-105, AGT-111 and AGT-112 gene with an effective amount of a modulator of AGT- 105, AGT-111 and AGT-112 expression for a time and under conditions sufficient to up- regulate or down-regulate or otherwise modulate expression o ⁇ AGT-105, AGT-111 and AGT-112.
  • a nucleic acid molecule encoding AGT-105, AGT-111 and AGT- 112 or a derivative or homolog thereof may be introduced into a cell to enhance the ability of that cell to produce AGT-105, AGT-111 and AGT-112, conversely, AGT-105, AGT-111 and AGT-112 sense and/or antisense sequences such as oligonucleotides may be introduced to decrease the availability of AGT-105, AGT-111 and AGT-112 molecules.
  • Another aspect of the present invention contemplates a method of modulating activity of AGT-105, AGT-111 and AGT-112 in a mammal, said method comprising administering to said mammal a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease AGT-105, AGT-111 and/or AGT-112 activity.
  • the molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of AGT-105, AGT-111 and AGT-112 or its ligand.
  • Modulating levels o ⁇ AGT-105, AGT-111 and/or AGT-112 expression or AGT-105, AGT- 111 and/or AGT-112 activity or function is important in the treatment of a range of conditions such as obesity, anorexia, energy imbalance, diabetes, metabolic syndrome, dyslipidemia, hypertension and insulin resistance. It may also be useful in the agricultural industry to assist in the generation of leaner animals, or where required, more obese animals. Accordingly, mammals contemplated by the present invention include but are not limited to humans, primates, livestock animals (e.g. pigs, sheep, cows, horses, donkeys), laboratory test animals (e.g. mice, rats, guinea pigs, hamsters, rabbits), companion animals (e.g. dogs, cats) and captured wild animals (e.g. foxes, kangaroos, deer). A particularly preferred host is a human, primate or livestock animal.
  • livestock animals e.g. pigs, sheep, cows, horses, don
  • the present invention contemplates therapeutic and prophylactic use of AGT- 105, AGT-111 and/or AGT-112 expression products or AGT-105, AGT-111 and AGT-112 genetic mutants and/or agonists or antagonists agents thereof.
  • the present invention contemplates, therefore, a method of modulating expression o ⁇ AGT- 105, AGT-111 and/or AGT-112 in a mammal, said method comprising contacting the AGT- 105, AGT-111 and/or AGT-112 genes with an effective amount of an agent for a time and under conditions sufficient to up-regulate, down-regulate or otherwise module expression o ⁇ AGT-105, AGT-111 and AGT-112.
  • Another aspect of the present invention contemplates a method of modulating activity of AGT-105, AGT-111 and/or AGT-112 in a subject, said method comprising administering to said subject a modulating effective amount of an agent for a time and under conditions sufficient to increase or decrease AGT-105, AGT-111 and/or AGT-112 activity or function.
  • Modulation of activity by the administration of an agent to a mammal can be achieved by one of several techniques, including, but in no way limited to, introducing into a mammal a proteinaceous or non-proteinaceous molecule which:
  • (iii) functions as an agonist of AGT-105, AGT-111 and/or AGT-112.
  • the molecules which may be administered to a mammal in accordance with the present invention may also be linked to a targeting means such as a monoclonal antibody, which provides specific delivery of these molecules to the target cells.
  • a further aspect of the present invention relates to the use of the invention in relation to mammalian disease conditions.
  • the present invention is particularly useful but in no way limited to use in a therapeutic or prophylactic treatment of obesity, anorexia, diabetes or energy imbalance.
  • another aspect of the present invention relates to a method of treating a mammal suffering from a condition characterized by one or more symptoms of obesity, anorexia, diabetes and/or energy imbalance, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the expression o ⁇ AGT-105, AGT-111 and/ 'or AGT-112 or sufficient to modulate the activity of AGT-105, AGT-111 and/or AGT-112.
  • the present invention relates to a method of treating a mammal suffering from a disease condition characterized by one or more symptoms of obesity, anorexia, diabetes or energy imbalance, said method comprising administering to said mammal an effective amount of AGT-105, AGT-111 and/or AGT-112 or AGT-105, AGT-111 and/or AGT-112.
  • An agent includes proteinaceous or non-proteinaceous molecules such as antibodies, natural products, chemical entities or nucleic acid molecules (including antisense molecules, sense molecules, ribozymes, ds-RNA molecules or DNA-targeting molecules).
  • an “effective amount” means an amount necessary at least partly to attain the desired immune response (e.g. against AGT-105, AGT-111 or AGT-112) or to delay the onset or inhibit progression or halt altogether the onset or progression of a particular condition.
  • AGT-105, AGT-111 and/or AGT-112 or AGT-105, AGT-111 and/or AGT-112 or agents capable of modulating the expression or activity of said molecules may be co-administered with one or more other compounds or other molecules.
  • co-administered is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes.
  • sequential administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules. These molecules may be administered in any order.
  • the present invention relates to the use of an agent capable of modulating the expression of AGT-105, AGT-111 and/or AGT-112 or a derivative, homolog or analog thereof in the manufacture of a medicament for the treatment of a condition characterized by obesity, anorexia, weight maintenance, diabetes and/or energy imbalance.
  • the present invention relates to the use of an agent capable of modulating the activity of AGT-105, AGT-111 and/or AGT-112 or a derivative, homolog, analog, chemical equivalent or mimetic thereof in the manufacture of a medicament for the treatment of a condition characterized by obesity, anorexia, weight maintenance, diabetes and/or energy imbalance.
  • a further aspect of the present invention relates to the use o ⁇ AGT-105, AGT-111 and/or AGT-112 or derivative, homolog or analog thereof or AGT-105, AGT-111 and/or AGT- 112 or derivative, homolog, analog, chemical equivalent or mimetic thereof in the manufacture of a medicament for the treatment of a condition characterized by obesity, anorexia, weight maintenance, diabetes and/or energy imbalance.
  • Still yet another aspect of the present invention relates to agents for use in modulating the expression of AGT-105, AGT-111 and/or AGT-112 or a derivative, homolog or analog thereof.
  • a further aspect relates to agents for use in modulating AGT-105, AGT-111 and/or AGT- 112 activity or a derivative, homolog, analog, chemical equivalent or mimetic thereof.
  • Still another aspect of the present invention relates to AGT-105, AGT-111 and/or AGT-112 or derivative, homolog or analog thereof or AGT-105, AGT-111 and/or AGT-112 or derivative, homolog, analog, chemical equivalent or mimetic thereof for use in treating a condition characterized by one or more symptoms of obesity, anorexia, weight maintenance, diabetes and/or energy imbalance.
  • the mammal undergoing treatment may be a human or an animal in need of therapeutic or prophylactic treatment.
  • the present invention contemplates in one embodiment a composition comprising a modulator o ⁇ AGT-105, AGT-111 and AGT-112 expression or AGT-105, AGT-111 and AGT-112 activity and one or more pharmaceutically acceptable carriers and/or diluents.
  • the composition comprises AGT-105, AGT-111 and AGT-112 or a derivative, homolog, analog or mimetic thereof and one or more pharmaceutically acceptable carriers and/or diluents.
  • the compositions may also comprise leptin or modulations of leptin activity or ob expression.
  • active components all such components of such a composition are referred to as "active components”.
  • compositions of active components in a form suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or other medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active components in the required amount in the appropriate solvent with optionally other ingredients, as required, followed by sterilization by, for example, filter sterilization, irradiation or other convenient means.
  • sterilization by, for example, filter sterilization, irradiation or other convenient means.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • AGT-105, AGT-111 and AGT-112 and AGT-105, AGT-111 and AGT-112 including AGT-105, AGT-111 and AGT-112 themselves are suitably protected, they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • compositions and preparations should contain at least 1% by weight of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
  • the amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ⁇ g and 2000 mg of active compound.
  • the tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and formulations.
  • Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
  • the principal active component may be compounded for convenient and effective administration in sufficient amounts with a suitable pharmaceutically acceptable carrier in dosage unit form.
  • a unit dosage form can, for example, contain the principal active component in amounts ranging from 0.5 ⁇ g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 ⁇ g to about 2000 mg/ml of carrier.
  • the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • effective amounts of AGT-105, AGT-111 and AGT-112 or AGT-105, AGT-111 and AGT-112 will range from 0.01 ng/kg/body weight to above 10,000 mg/kg/body weight. Alternative amounts range from 0.1 ng/kg/body weight to above 1000 mg/kg/body weight.
  • the active ingredients may be administered per minute, hour, day, week, month or year depending on the condition being treated.
  • the route of administration may vary and includes intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, via suppository, via infusion, via drip, orally or via other convenient means.
  • the pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating AGT-105, AGT-111 and AGT-112 expression or AGT-105, AGT-111 and AGT-112 activity.
  • the vector may, for example, be a viral vector.
  • Still another aspect of the present invention is directed to antibodies to AGT-105, AGT- 111 and AGT-112 and their derivatives and homologs insofar as AGT-105, AGT-111 and
  • AGT-112 are proteins. Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to AGT-105, AGT-111 and AGT-112 or may be specifically raised to AGT-105, AGT-111 and AGT-112 or derivatives or homologs thereof. In the case of the latter, AGT-105, AGT-111 and AGT-112 or their derivatives or homologs may first need to be associated with a carrier molecule.
  • the antibodies and/or recombinant AGT-105, AGT-111 and AGT-112 or their derivatives of the present invention are particularly useful as therapeutic or diagnostic agents.
  • AGT-105, AGT-111 and AGT-112 and their derivatives can be used to screen for naturally occurring antibodies to AGT-105, AGT-111 and AGT-112 which may occur in certain autoimmune diseases or where cell death is occurring. These may occur, for example, in some autoimmune diseases.
  • specific antibodies can be used to screen for AGT-105, AGT-111 and AGT-112. Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA.
  • Antibodies to AGT-105, AGT-111 and AGT-112 of the present invention may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to the AGT-105, AGT-111 and AGT-112 or may be specifically raised to the AGT-105, AGT- 111 and AGT-112 or their derivatives. In the case of the latter, the AGT-105, AGT-111 and AGT-112 protein may need first to be associated with a carrier molecule. Alternatively, fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies. The antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool or as a means for purifying AGT-105, AGT-111 and AGT-112.
  • specific antibodies can be used to screen for AGT-105, AGT-111 and AGT- 112 proteins.
  • the latter would be important, for example, as a means for screening for levels of AGT-105, AGT-111 and AGT-112 in a cell extract or other biological fluid or purifying AGT-105, AGT-111 and AGT-112 made by recombinant means from culture supernatant fluid.
  • Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.
  • any second antibodies (monoclonal, polyclonal or fragments of antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody.
  • An antibody as contemplated herein includes any antibody specific to any region of AGT-105, AGT-111 and AGT-112.
  • Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utilizable for immunoassays.
  • the methods of obtaining both types of sera are well known in the art.
  • Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of AGT-105, AGT-111 and AGT-112, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques.
  • antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
  • the use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example, Basic Facts about Hybridomas, in Compendium of Immunology Vol. II, ed. by Schwartz, 1981; Kohler and Milstein, Nature 256: 495-499, 1975; Kohler and Milstein, European Journal of Immunology 6: 511-519, 1976).
  • Another aspect of the present invention contemplates a method for detecting AGT-105, AGT-111 and AGT-112 or a derivative or homolog thereof in a biological sample from a subject, said method comprising contacting said biological sample with an antibody specific for AGT-105, AGT-111 and AGT-112 or their antigenic derivatives or homologs for a time and under conditions sufficient for a complex to form, and then detecting said complex.
  • the presence of the complex is indicative of the presence of AGT-105, AGT-111 and AGT-112.
  • This assay may be quantitated or semi-quantitated to determine a propensity to develop obesity or other conditions or to monitor a therapeutic mitum.
  • AGT-105, AGT-111 and AGT-112 may be accomplished in a number of ways such as by Western blotting and ELISA procedures.
  • a wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These, of course, includes both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.
  • Sandwich assays are among the most useful and commonly used assays. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule.
  • a second antibody specific to the AGT-105, AGT-111 and AGT-112, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-AGT-105, AGT-111 and AGT-112-labelled antibody. Any unreacted material is washed away, and the presence of AGT-105, AGT-111 and AGT-112 is determined by observation of a signal produced by the reporter molecule.
  • the results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten.
  • Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent.
  • the sample is one which might contain AGT-105, AGT-111 and AGT-112 including cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid.
  • the sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex to the solid surface which is then washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g. from room temperature to about
  • the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of AGT-105, AGT-111 and AGT-112.
  • the second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to AGT-105, AGT-111 and AGT-112.
  • An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
  • reporter molecule as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
  • an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
  • glutaraldehyde or periodate As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, ⁇ -galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase.
  • fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above.
  • the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen- antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample.
  • a "reporter molecule” also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody absorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
  • the fluorescent-labelled antibody is allowed to bind to the first antibody- hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength. The fluorescence observed indicates the presence of the hapten of interest.
  • Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
  • the present invention also contemplates genetic assays such as involving PCR analysis to detect AGT-105, AGT-111 and AGT-112 or their derivatives.
  • the assays of the present invention may also extend to measuring AGT-105, AGT-111 and AGT-112 or AGT-105, AGT-111 and AGT-112 in association with ob or leptin.
  • a Psammomys obesus colony is maintained at Deakin University, with the breeding pairs fed ad libitum a diet of lucerne and chow.
  • Experimental animals were weaned at four weeks of age and given a diet of standard laboratory chow from which 12% of energy was derived from fat, 63% from carbohydrate and 25% from protein (Barastoc, Pakenham, Australia). Animals were housed individually in a temperature controlled room (22 ⁇ 1°C) with a 12- 12-hour light-dark cycle. At 18 weeks of age, animals were sacrificed and the tissues immediately removed, frozen in liquid N and then stored at -80°.
  • Psammomys obesus can be classified into three groups according to their blood glucose and plasma insulin concentration, taken in the fed state at 16 weeks of age.
  • Group A animals are normoglycemic (blood glucose ⁇ 8.0 mmol/L) and normoinsulinemic (plasma insulin ⁇ 150 mU/L)
  • Group B animals are normoglycemic but hyperinsulinemic (plasma insulin >150 mU/I)
  • Group C animals are hyperglycemic (blood glucose >150 mU/I) and hyperinsulinemic.
  • AGT-105, AGT-111 and AGT-112 were all identified by differential display PCR using the RNAimage mRNA differential display system (GenHunter Corporation). Hypothalamus RNA from fed and fasted, lean and obese Psammomys obesus was compared. The PCR products were separated on a 4.5% w/v polyacrylamide gel and differentially expressed PCR fragments were visualized by exposing the dried gel to x-ray film. Candidate bands were excised from the gel and reamplified by PCR using the appropriate primer combination. Sequencing reactions were carried out using ABI PRISM Big-Dye terminator cycle sequencing ready reaction kits and analyzed on an ABI 373 DNA sequencer.
  • Plasma insulin concentrations were determined using a double antibody solid phase radioimmunoassay (Phadeseph, Kabi Pharmacia Diagnostics, Sweden).
  • Oligonucleotide primers for the AGT-105, AGT-111 and AGT-112 gene PCR were chosen from the sequence previously determined. Primers were also designed to the Psammomys obesus ⁇ -actin gene to use as a housekeeping gene.
  • AGT-111 5 ' -CATGTGTTCCCTTCATGCACTGATCTGT-3 ' [SEQ ID NOT0]
  • AGT-105 5 ' -CTTGCAGAGGACAACTCACATGGTAAACACA-3 ' [SEQ ID NOT 1]
  • ⁇ -actin gene 5 ' -TCCGGTCCACAATGCCTGGGTACAT- 3 ' [SEQ ID NO: 12] All probes had the reporter dye FAM attached to the 5' end and the quencher dye TAMRA attached to the 3' end. PCR conditions were 50°C for 2 min, 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min.
  • AGT-112 Gene expression of AGT-112 in each cDNA sample was quantitated using SYBR Green PCR on an ABI Prism 7700 sequence detector, ⁇ -actin was used as the endogenous control to standardize the amount of cDNA added to a reaction, as for AGT-111 and AGT-105. Primer sequences were as follows:
  • PCR conditions were 50°C for 2 min, 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min.
  • hypothalamic mRNA profile of lean, obese and diabetic Psammomys obesus in the fed and fasted state compared the hypothalamic mRNA profile of lean, obese and diabetic Psammomys obesus in the fed and fasted state. cDNA fragments amplified were found to be differentially expressed between groups. EXAMPLE 9 AGT-105 gene expression in the hypothalamus
  • AGT-105 gene expression was increased in the hypothalamus of Group A animals compared to Group B or C animals. There was a negative correlation with body weight (with all animals together p ⁇ 0.02) and percent body fat (with all animals together p ⁇ 0.01 and in Group A animals alone, p ⁇ 0.05). There was also a negative correlation with log insulin (with all animals p ⁇ 0.05).
  • the partial nucleotide sequence o ⁇ Psammomys obesus AGT-105 cDNA is as follows:-
  • the nucleotide sequence does not match any known genes that are currently in the GenBank database.
  • Taqman real time PCR was used to determine the tissue distribution o ⁇ AGT-105 gene expression. Expression was found in all regions of the brain analyzed (hypothalamus, brain stem, cerebellum, cortex, hippocampus and midbrain). A low level of expression was also seen in the testes. No expression at all was found in skeletal muscle, adipose tissue, liver, heart, kidney or ovaries.
  • AGT-111 gene expression in the hypothalamus is seen to increase significantly with fasting in all groups. There is a significant negative correlation with log glucose in all animals and separately in both the fed and fasted state. There is also a negative correlation with log insulin when all animals are analyzed together.
  • This sequence does not match any known gene or ESTs in the GenBank database.
  • Taqman real time PCR was used to determine the tissue distribution o ⁇ AGT-111 gene expression. Expression was highest in the brain regions (hypothalamus, brain stem, cerebellum, cortex, hippocampus and midbrain) and testes, but also found in skeletal muscle, adipose tissue, liver, heart, kidney or ovaries.
  • AIHW Australian Institute of Health and Welfare
  • Heart Stroke and Vascular diseases
  • AIHW Cat. No. CVD 7 Australia AIHW and the Heart Foundation of Australia, 1999.
  • AIHW Australian Institute of Health and Welfare

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Abstract

La présente invention concerne généralement des molécules d'acide nucléique exprimées au moins dans l'hypothalamus, identifiées au moyen de techniques de criblage différentiel dans différents états physiologiques. Les molécules d'acide nucléique sont associées à ou agissent comme des marqueurs pour des états de bonne santé, d'obésité, d'anorexie, de maintien de poids, de diabète et/ou des niveaux d'énergie métabolique. L'invention concerne plus particulièrement une molécule d'acide nucléique et/ou son produit d'expression destinés à être utilisés dans des protocoles thérapeutiques ou diagnostiques pour des états tels que l'obésité, l'anorexie, le maintien de poids, le diabète et le déséquilibre énergétique. La molécule d'acide nucléique, le produit d'expression et leurs dérivés, homologues, analogues et mimétiques conviennent, par conséquent, comme agents thérapeutiques ou diagnostiques concernant l'obésité, l'anorexie, le maintien de poids, le diabète et le déséquilibre énergétique, ou comme cibles destinées à la conception et/ou l'identification de modulateurs de leur activité et/ou fonction.
PCT/AU2002/001099 2001-08-14 2002-08-13 Genes lies a l'obesite exprimes au moins dans l'hypothalamus WO2003016542A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2004063218A1 (fr) * 2003-01-13 2004-07-29 Autogen Research Pty Ltd Genes relatifs a l'obesite

Citations (1)

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WO1999023217A1 (fr) * 1997-10-31 1999-05-14 International Diabetes Institute Nouveau gene et ses utilisations

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999023217A1 (fr) * 1997-10-31 1999-05-14 International Diabetes Institute Nouveau gene et ses utilisations

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Title
KORNER J. ET AL.: "Effects of leptin receptor mutation on Agrp gene expression in fed and fasted lean and obese (LA/Nfaf) rats", ENDOCRINOLOGY, vol. 141, no. 7, 2000, pages 2465 - 2471 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004063218A1 (fr) * 2003-01-13 2004-07-29 Autogen Research Pty Ltd Genes relatifs a l'obesite

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